Jurkat cells were added without or with different ligands for 24 h and then incubated in 500 μL binding buffer, followed by 10 μL allophycocyanin-conjugated Annexin V and PI (BD Pharmingen) treatment away from light for 15 min. The samples were detected by flow cytometry (FACS Caliber, BD Bioscience, San Diego, CA, USA) immediately.
Annexin 5 and pi
Manufactured by BD
Sourced in United States
Annexin V and PI are laboratory reagents used in cell biology and biochemistry research. Annexin V is a protein that binds to phosphatidylserine, a lipid found on the surface of cells undergoing apoptosis (programmed cell death). PI (Propidium Iodide) is a fluorescent dye that intercalates with DNA, allowing the detection of dead or dying cells. These reagents are commonly used together in flow cytometry and microscopy techniques to analyze cell viability and apoptosis.
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Lab products found in correlation
Cytomics fc 500 mpl, by Beckman Coulter (3 mentions)
Facscalibur, by BD (3 mentions)
Annexin 5, by BD (2 mentions)
Facscalibur flow cytometer, by BD (2 mentions)
Facs caliber, by BD (1 mentions)
Cxp software, by Beckman Coulter (1 mentions)
Cytomics fc500, by Beckman Coulter (1 mentions)
Anti cd69 mab, by BD (1 mentions)
Anti mouse cd3 mab, by BioLegend (1 mentions)
Mouse il 2, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (1 mentions)
Macsquant vyb, by Miltenyi Biotec (1 mentions)
B220 fitc, by Thermo Fisher Scientific (1 mentions)
Fixation permeabilization concentrate and diluent kit, by Thermo Fisher Scientific (1 mentions)
Accuri c6, by Tree Star (1 mentions)
Facsaria, by Tree Star (1 mentions)
Facscanto, by BD (1 mentions)
Plate coated anti btla, by Thermo Fisher Scientific (1 mentions)
Sp6800, by Sony (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
32 protocols using annexin 5 and pi
1
Apoptosis Assay in Jurkat Cells
2
Annexin V-PI Apoptosis Assay Protocol
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PBMCs (2 × 106/mL) were remained untreated or treated with TSA (300 nmol) for 24 h. The cells were washed in cold phosphate-buffered saline (1x PBS), the supernatant was discarded, and 106 PBMCs were resuspended in 1 mL 1x annexin binding buffer. 5 μL of annexin V and PI (BD Pharmingen™, San Diego, CA) were incubated in 100 μL of resuspended PBMCs. After 15 min incubation at room temperature in the dark, 400 μL of 1x annexin binding buffer was added and cells were acquired by a flow cytometer (Cytomics FC 500; Beckman Coulter, Fullerton, CA, USA). Results were analyzed and presented with CXP software (Beckman Coulter).
3
Characterizing CD4+ T Cell Activation
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CD4+ T cells were isolated as described above according to the manufacturer’s instructions, then, 4 × 105 of the purified T cells were seeded in 96-well round-bottomed microtiter plates precoated overnight with 5 µg/ml of anti-mouse CD3 mAb (Biolegend, CA, USA) in the presence of 1 µg/ml of anti-mouse CD28 mAb and 1,000 U/ml of mouse IL-2 (Peprotech, Rocky Hill, NJ, USA).
For the activation assay, T cells were collected and stained with a PE-labeled anti-CD69 mAb for 5 h. For the T cell proliferation assay, 10 nM BrdU was added to the plates for 48 h, then incorporated BrdU was detected using a BrdU ELISA kit (BD Biosciences, San Jose, CA, USA) after DNase treatment according to the manufacturer’s protocol. For the apoptosis assay, cells were collected and stained with Annexin V and PI as recommended by the manufacturer (BD Biosciences, San Jose, CA, USA).
For the activation assay, T cells were collected and stained with a PE-labeled anti-CD69 mAb for 5 h. For the T cell proliferation assay, 10 nM BrdU was added to the plates for 48 h, then incorporated BrdU was detected using a BrdU ELISA kit (BD Biosciences, San Jose, CA, USA) after DNase treatment according to the manufacturer’s protocol. For the apoptosis assay, cells were collected and stained with Annexin V and PI as recommended by the manufacturer (BD Biosciences, San Jose, CA, USA).
4
Detecting Apoptosis Using Annexin-V and PI
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To detect apoptosis, cells were incubated with RY-2f or DMSO at different concentrations for 24 h. The cells were harvested, washed twice with cold 1 × PBS, and re-suspended in 200 μL binding buffer at density of 1 × 105 cells / mL. The cells were then stained with 5 μL Annexin-V and PI (BD Biosciences) for 15 min in dark condition at room temperature and subjected to analysis by flow cytometry (Cytomics FC 500 MPL, Beckman Coulter). The early apoptosis was evaluated based on the percentage of cells with Annexin V+/PI−, while the late apoptosis was that of cells with Annexin V+/PI+. The results were indicated as mean values from three independent determinations.
5
Evaluating Exosome Effects on Dendritic Cell Apoptosis
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BMDCs were generated from bone marrow cells isolated from C57BL/6 mice, as previously described [17 (link)]. Day 8 immature BMDCs were incubated with lipopolysaccharide (LPS, 100 ng/mL; Invitrogen, San Diego, CA, USA) or exosomes (5 and 25 μg/mL) isolated from non-irradiated (N-exo) and gamma-irradiated cancer cells (G-exo) for 18 h in a humidified incubator containing 5% CO2 at 37 °C. Cells were then harvested and stained with Annexin V and PI (BD Bioscience) according to the systematic instructions provided by the manufacturer. Samples (stained cells) were analyzed using a FACS instrument (MACSQuant VYB; Miltenyi Biotec, San Diego, CA, USA).
6
Comprehensive Immune Cell Profiling
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Lymphocytes from each organ was stained with various combinations of mAbs to CD4-FITC/APC, CD8α-PE/APC, CD3α-PE, and B220-FITC (eBiosciences), CD103-APC, LAP (TGF-β1)-PE (Clone TW7-20B9, Biolegend). Fixation/Permeabilization Concentrate and Diluent Kit (eBiosciences) were used for the intracellular staining of FoxP3-PE/APC according to the user’s handbook. After stained with surface markers, cells were stained with Annexin V and PI (BD Pharmingen) to examine apoptosis. For in vivo BrdU incorporation assay, mice were administered of BrdU (16 mg/ml; 50 μl) via i.n. route [48 (link)]; cells were harvested after 24hours and stained with BD FastImmune BrdU kit (BD Biosciences). Samples were run on BD Biosciences FACSCalibur, FACSAria or Accuri C6 instruments, and analyzed with FlowJo software (Tree Star, Ashland, OR).
7
Annexin V/PI Apoptosis Assay
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Cells were seeded in 12-well plates at a density of 1 × 105 cells/well. After 24 h, cells were washed with ice-cold DPBS plus 5 mM EDTA and stained with annexin V and PI (BD Biosciences) for 15 min at RT. Analysis was carried out using FACS Canto (BD Biosciences).
8
BTLA Modulation of PBMC Apoptosis
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Untreated control PBMC or PBMC knocked down by shRNA from healthy donors were treated in 96-well plates with plate-coated anti-BTLA (10 μg/mL; eBioscience, San Diego, CA) or PBS for 1 d, washed with PBS followed by co-staining with Annexin V and PI (BD Bioscience). The apoptosis rate was examined using flow cytometry.
9
Vernodalol-induced Apoptosis Analysis
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The cells were cultured (5×105) in each well of 6-well plates to 70–80% confluence with RPMI-1640 and then treated with vernodalol at various concentrations (0, 50, 75 or 100 µM) for the indicated time (24 h). The vernodalol-treated and control cells (treated with DMSO only) were harvested by centrifugation for 5 min at 377 × g and room temperature and washed twice with cold 1X PBS. The cells were then stained with 5 µl Annexin V and PI (BD Biosciences) for 15 min in dark conditions at room temperature according to the manufacturer's protocol and then subjected to analysis by flow cytometry (FACS Calibur; BD Biosciences). Data were analyzed using Flowjo software version 10.0 (Tree Star, Inc., Ashland, OR, USA). Early apoptosis was evaluated based on the percentage of cells with Annexin V+/PI− staining, and late apoptosis was evaluated based on the percentage of cells with Annexin V+/PI+ staining.
10
Cell Cycle and Apoptosis Analysis
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The HOS and MG-63 cells, transiently transfected with si-Xist or si-NC, were collected 48h after transfection by trypsinization. For cell cycle analysis, OS cells were first fixed using 70% ethanol and stored at 4 °C overnight. Followed by the next day, the OS cells were washed with PBS, treated with 50 mg/ml RNase A and stained with 50 mg/ml propidium iodide (PI) for 30 min in the dark. Cell cycle distribution were analyzed by flow cytometry (FACSCalibur, Becton-Dickinson). For apoptosis assay, cells were harvested and were resuspended in 1× binding buffer. After double staining with Annexin V and PI (BD Pharmingen, USA), cells were analyzed according to manufacturer's instructions to detect early and late apoptosis of cells. All the samples were assayed in triplicate.
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